Project Summary Central neurons are highly specialized and long-lived cells that form precise circuitry to support normal brain function. The cerebral cortex does not add new neurons to maintain or increase function nor replace neurons lost to injury or disease. Adult hippocampal neurogenesis in the human brain shows that adult neurogenesis is possible. However, for human cerebral cortex, there are two possible strategies for neuronal addition, 1) replace with exogenous neurons generated by cell culture or 2) recruit local endogenous cells by converting them to neurons. The ultimate goal of this project is to investigate the second approach and to establish the capacity for neuronal reprogramming of human glial progenitor cells and to assess their potential for functional integration. For neuronal reprogramming to have therapeutic potential, it will be necessary to precisely engineer neurons with a predictable and sustainable rate of survival. Aim 1 will address issues of survival efficiency and subtype precision, developing an innovative vector toolbox to conduct these studies. But there also remains the question of how authentic these induced neurons become. The forced transcription factor expression may cause expression of certain neuronal phenotypes, including the capacity to fire an action potential, without necessarily resulting in functional maturation. While our preliminary data demonstrate the most advanced neuronal morphology reported to date, true circuit integration still remains to be shown. Aim 2 is designed to utilize current advances in connectivity tracing to investigate the state of integration of newly engineered neurons into local and distant circuits. In particular Aim 2b will ask if newly engineered neurons are capable of actively rewiring in the brain. We see application for this approach in rebuilding any damaged circuit, including ultimately those experiencing dysregulation such as in epilepsy or neuropathic pain. There is no information about the capacity to reprogram non-neuronal cells in the human brain into new neurons, despite the compelling need for such data to advance therapeutic application of this approach. As we cannot conduct these experiments in human brain, Aim 3 will develop a chimeric model, engrafting human cells into rodent brain to allow targeting of human glial progenitor cells for neuronal reprogramming and evaluating the extent of circuit integration with rat neurons. By using human iPSC-derived glial progenitor cells as our starting population, it will be possible to expand these studies to address the impact of disease-specific factors as well as providing a basis for eventual patient-specific therapies.